Measurement of disease marker substances (metabolites, proteins, cells etc.) present in low concentrations in a body fluid (blood, urine etc.) is generally carried out by employing biological reactions such as enzymatic reaction and antigen-antibody binding. Since enzyme and antibody exhibit very high reaction specificities of selectively recognizing their reaction partners and high reaction efficiencies, determination of analytes in a complex medium becomes possible. It is very important to seek development of diagnostic system based on such reaction characteristics so that early diagnosis of disease and adequate treatment of the disease in its early stage may be conducted. However, as most of the diagnostic systems require handling of reagents and devices, their use is limited to laboratories and further expert knowledge is necessary for conducting the testing.
Recently, as a category of immunoassay, the need of self-diagnosis at home as well as testing at the point of care such as doctor's office or emergency room for marker substances such as hormones, proteins and microorganisms that may indicate symptoms and progress of disease, is rapidly growing (Reference: C. P. Price et al., Principles and Practice of Immunoassay, 1997, page 579-603, Macmillan Reference Ltd., London). To this end, development of immunoassay system that does not require any expert knowledge and complex procedure, is simple to use, and provides quick response has been necessary. Such diagnostic performances could be achieved by an immuno-chromatography method that employs a microporous membrane for immobilizing a binding protein (e.g. antigen or antibody) (Reference: R. Chen et al., 1987, Clin. Chem. Vol. 33, Page 1521-1525; M. P. A. Laitinen, 1996, Biosens, Bioelectron., Vol. 11. 1207-1214: S. C. Lou et al., 1993, Clin. Chem., Vol. 39, 619-624; S. H. Paek et al., 1999, Anal. Lett., Vol. 32. 335-360). In this analytical format, when analyte-containing specimen is absorbed from the bottom end of the membrane strip, the analyte is transported to the layer of immobilized binding protein by the capillary action through membrane pores. A binding reaction between the antigen and antibody occurs on the surface of solids, and unbound molecules are subsequently separated by the medium flow. As the transfer of the reactant is accelerated by the lateral flow of medium, the membrane strip immuno-chromatography method based on the above principle provides a quick analysis of analyte and convenience of one-step detection where the analysis can be completed upon sample application alone.
The demand for such one-step diagnostic system has been well reflected in rapid growth of market of diagnostic kits for pregnancy and ovulation, and as establishment of internet-based telediagnosis and prescription system is soon expected, home monitoring system of diseases such as adult disease requiring periodical examination will be required as a key element of health care. However, current home-version diagnostic reagents are mostly at level of performing simple immuno-chromatographic assay and identifying the qualitative result of a color signal with naked eye, and thus unsuitable for analysis of indicator substances (protein marker etc.) requiring the determination of their concentrations. As conventional method that can be used for quantitative analysis, the color signal generated from gold colloids used as tracer can be converted to optical density using a conventional photometric transducing means (Reference: M. P. A. Laitinen, 1996, Biosens. Bioelectron., Vol. 11, 1207-1214), yet it has disadvantage, i.e. poor detection sensitivity in comparison to that of an enzyme-linked immunosorbent assay widely used in laboratories.
The disadvantage of low sensitivity from the point-of-care testing device can be overcome by using signal generator with high sensitivity such as fluorescent substance or radioisotope. Actually, an immunoassay system was developed, where an immuno-chromatographic assay was performed using a detection antibody labeled with fluorescent substance and the assay result was measured with a fluorescence detector (Reference: U.S. Pat. No. 5,753,517). As this technique provided high sensitivity as well as no harmful effect, it has recently been applied to a point-of-care immunodiagnostic device that can be used in emergency room. (Reference: U.S. Pat. No. 6,271,040 B1). However, because fluorescence detectors are relatively too expensive and difficult to be reduced to a portable size, the system could be limitedly used in clinical laboratory of hospital or research laboratory, and thus when comparing with laboratory-version enzyme linked immunosorbent assay, there is no special advantage other than quick assay.
On the other hand, laboratory-version enzyme-linked immunosorbent assay essentially requires washing procedure for separating immune complexes from unreacted substance in the respective process of immunoassay, and further should carry out, separately, enzymatic reaction for signal generation. Consequently, such complex, multi-step procedure is clearly difficult to use for point-of-care testing.
The present invention makes it the object to provide a membrane strip biosensor technique which not only enables quick and simple assay required in point-of-care testing but also satisfies clinical needs for highly sensitive determination of analytes in specimen by applying to point-of-care testing the principle of laboratory-version enzyme-linked immunosorbent assay which provides advantage, being relatively cheap and high sensitivity.